Related Research Articles
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net electrical charge or is electrically neutral in the statistical mean. The standard nomenclature to represent the isoelectric point is pH(I). However, pI is also used. For brevity, this article uses pI. The net charge on the molecule is affected by pH of its surrounding environment and can become more positively or negatively charged due to the gain or loss, respectively, of protons (H+).
A monoclonal antibody is an antibody produced from a cell lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.
A polyhistidine-tag, best known by the trademarked name His-tag, is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as a hexa histidine-tag, 6xHis-tag, or His6 tag. The tag was invented by Roche, although the use of histidines and its vectors are distributed by Qiagen. Various purification kits for histidine-tagged proteins are commercially available from multiple companies.
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants won't interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
An ion-exchange resin or ion-exchange polymer is a resin or polymer that acts as a medium for ion exchange. It is an insoluble matrix normally in the form of small microbeads, usually white or yellowish, fabricated from an organic polymer substrate. The beads are typically porous, providing a large surface area on and inside them where the trapping of ions occurs along with the accompanying release of other ions, and thus the process is called ion exchange. There are multiple types of ion-exchange resin. Most commercial resins are made of polystyrene sulfonate, followed up by polyacrylate.
Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.
Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.
Ion exchange is a reversible interchange of one kind of ion present in an insoluble solid with another of like charge present in a solution surrounding the solid with the reaction being used especially for softening or making water demineralised, the purification of chemicals and separation of substances.
Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.
Sephadex is a cross-linked dextran gel used for gel filtration. It was launched by Pharmacia in 1959, after development work by Jerker Porath and Per Flodin. The name is derived from separation Pharmacia dextran. It is normally manufactured in a bead form and most commonly used for gel filtration columns. By varying the degree of cross-linking, the fractionation properties of the gel can be altered.
Solid-phase extraction (SPE) is a solid-liquid extractive technique, by which compounds that are dissolved or suspended in a liquid mixture are separated, isolated or purified, from other compounds in this mixture, according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue.
Fast protein liquid chromatography (FPLC) is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid (the mobile phase) and a porous solid (the stationary phase). In FPLC the mobile phase is an aqueous buffer solution. The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.
Hydrophilic interaction chromatography is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.
Depyrogenation refers to the removal of pyrogens from solutions, most commonly from injectable pharmaceuticals.
DNA separation by silica adsorption is a method of DNA separation that is based on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH conditions.
Chromatography is a physical method of separation that distributes the components you want to separate between two phases, one stationary, the other moving in a definite direction. Cold ethanol precipitation, developed by Cohn in 1946, manipulates pH, ionic strength, ethanol concentration and temperature to precipitate different protein fractions from plasma. Chromatographic techniques utilise ion exchange, gel filtration and affinity resins to separate proteins. Since the 1980s it has emerged as an effective method of purifying blood components for therapeutic use.
Diethylaminoethyl cellulose (DEAE-C) is a positively charged resin used in ion-exchange chromatography, a type of column chromatography, for the separation and purification of proteins and nucleic acids. Gel matrix beads are derivatized with diethylaminoethanol (DEAE) and lock negatively charged proteins or nucleic acids into the matrix. The proteins are released from the resin by increasing the salt concentration of the solvent or changing the pH of the solution as to change the charge on the protein.
Chromatofocusing is a protein-separation technique that allows resolution of single proteins and other ampholytes from a complex mixture according to differences in their isoelectric point. Chromatofocusing utilizes ion exchange resins and is typically performed on fast protein liquid chromatography (FPLC) or similar equipment capable of producing continuous buffer gradients though this is not a requirement. In contrast to typical ion exchange chromatography, where bound molecules are eluted from the resin by increasing the ionic strength of the buffer environment, chromatofocusing elutes bound species by altering the pH of the buffer. This changes the net surface charge of bound molecules, altering their avidity for the resin. As the changing pH of the buffer system traverses the pI of a given molecule, that molecule will elute from the resin as it will no longer possess a net surface charge. Chromatofocusing is a powerful purification technique with respect to proteins as it can resolve very similar species only differing by 0.02 pH units that may not separate well, or at all, using traditional ion exchange strategies. A major drawback to this technique is that some proteins will aggregate when they are present at relatively high concentrations and carry no net surface charge. This can cause blockage of the resin, which is highly problematic when using sealed columns of ion exchange resin on FPLC equipment, resulting in pressure buildup and possible equipment failure. Apparent aggregation issues can sometimes be overcome by limiting the sample concentration and use of buffer additives that deter aggregate formation.
Lysozyme PEGylation is the covalent attachment of Polyethylene glycol (PEG) to Lysozyme, which is one of the most widely investigated PEGylated proteins.
References
- ↑ Lee, Y.C. (1990). "High-performance Anion-exchange Chromatography for Carbohydrate Analysis". Analytical Biochemistry. 189 (2): 151–62. doi: 10.1016/0003-2697(90)90099-u . PMID 2281856.
- 1 2 Fotsis, T., H. Adlercreutz; Järvenpää, Paula; Setchell, K.D.R.; Axelson, M.; Sjövall, J. (1981). "Group Separation of Steroid Conjugates by DEAE-Sephadex Anion Exchange Chromatography". Journal of Steroid Biochemistry. 14 (5): 457–63. doi:10.1016/0022-4731(81)90357-5. PMID 7300338.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ↑ Rocklin, Roy D.; Pohl, Christopher A. (1983). "Determination of Carbohydrates by Anion Exchange Chromatography with Pulsed Amperometric Detection". Journal of Liquid Chromatography. 6 (9): 1577–590. doi:10.1080/01483918308064876.
- ↑ Duong-Ly, Krisna C.; Gabelli, Sandra B. (2014). "Using Ion Exchange Chromatography to Purify a Recombinantly Expressed Protein". Laboratory Methods in Enzymology: Protein Part C. Vol. 541. Academic Press. pp. 95–103. doi:10.1016/b978-0-12-420119-4.00008-2. ISBN 9780124201194. PMID 24674065.
- ↑ "FPLC Ion Exchange and Chromatofocusing: Principles and Methods". Pharmacia Biotech. Pharmacia Biotech AB. 1985.
- ↑ Guide to Ion-Exchange Chromatography. Harvard Apparatus. p. 2.
- ↑ Guide to Ion-Exchange Chromatography. Harvard Apparatus. p. 3.